The principal goal of the proposed research is to reach an understanding of the mechanism of Saccharomyces cerevisiae DNA replication using a combination of biochemical and genetic techniques. The major emphasis of the research will be on the study of the yeast replication fork, its formation, its structure and properties, and on the biochemical and genetic characterization of several of its components. A clear understanding of the yeast replication fork is important in realizing our second major objective, which is to reconstitute origin specific DNA replication in a (partially) purified in vitro system. The interactions between DNA polymerases delta and epsilon and their accessory factors will be studied biochemically using various model replication systems. Genetic studies will focus on cloning the genes encoding the accessory factors and on the generation and study of temperature sensitive mutants in some of these genes. Replicational DNA helicases will be purified and studied in replication fork model assays. The participation of the DNA polymerases in the formation of the replication fork will be studied. We will attempt to establish origin specific DNA replication in an in vitro reconstitution assay providing known elongation proteins in purified form and assaying for additional factor(s) necessary for origin-specific DNA replication. The required factor(s) will then be further purified and studied in detail. The proposed study of eukaryotic DNA replication will focus on yeast because of all eukaryotic organisms yeast is most amenable to genetic and biochemical manipulation. An additional impetus for studying yeast as an eukaryotic model system follows from recent results which show that yeast and mammalian replication proteins are structurally and functionally remarkably homologous. Thus, an intensive study of the mechanism of DNA replication and its regulation in yeast will aid in understanding the mechanisms that induce or prevent mutations as well as those that control cell division in human cells.